The detection and quantification of endotoxin (lipopolysaccharide, LPS) is critically important in a wide range of health-related contexts, including human healthcare, clinical and basic medical research, pharmaceutical manufacturing, occupational and public health and food and water purity testing. Currently, most endotoxin detection or quantification methods are based on the Limulus Amoebocyte Lysate-related gelation reaction or chromogenic response as modified from the original Limulus Amoebocyte Lysate Assay (LAL Assay) first reported in 1960s.
The Limulus Amoebocyte is the only circulating cell found in the blood of Limulus polyphemus, the horseshoe crab. When a horseshoe crab acquires a Gram-negative bacterial infection, the Limulus Amoebocyte Lysate enzyme interacts with the Lipid A portion of the LPS produced and triggers extracellular coagulation. This reaction is the basis of a number of assay methods used for detecting and quantifying endotoxin in aqueous specimens (e.g., kinetic turbidimetric LAL assay, kinetic chromogenic LAL assay, Gel-Clot LAL, and End-Point LAL), and endotoxin detection limits using these assays can be as low as the pg/mL range.
However, the current LAL-based assays have a number of disadvantages. For example, LPS isolated from different species of bacteria do not activate LAL equally. In addition, certain substances interfere with LAL's ability to react with endotoxin. Furthermore, since the lysate is a crude and variable mixture, not a single purified enzyme, the enzyme activity needs to be standardized for every batch of LAL extracted using a complex and expensive procedure. The reagents for LAL assays are also derived from animals, and the reagents need to be stored under controlled conditions, such as controlled temperature. In general, the complexity of the assays requires the use of skilled technicians. The limitations of current assays for LPS demonstrate a continuing need for a simple and low cost, yet rapid, sensitive and selective, assay for reporting and quantifying LPS in aqueous samples.
Previously, assay devices that employ liquid crystals as a means to detect and quantify various analytes have been disclosed. For example, a liquid crystal assay device using mixed self-assembled monolayers (SAMs) containing octanethiol and biotin supported on an anisotropic gold film obliquely deposited on glass has been reported. Gupta, V. K.; Skaife, J. J.; Dubrovsky, T. B., Abbott N. L. Science, 279, (1998), pp. 2077-2079. In addition, PCT publication WO 99/63329 published on Dec. 9, 1999, discloses assay devices using SAMs attached to a substrate and a liquid crystal layer that is anchored by the SAM. U.S. Pat. No. 6,288,392 issued to Abbott et al. discloses the quantitative characterization of obliquely-deposited substrates of gold using atomic force microscopy and describes the influence of substrate topography on the anchoring of liquid crystals. U.S. Pat. No. 6,284,197 issued to Abbott et al. discloses the optical amplification of molecular interactions using liquid crystals.
Past studies have also reported on the influence of surfactants on the orientations of liquid crystals when the surfactants are adsorbed at interfaces of aqueous phases and thermotropic liquid crystals in emulsions (Drzaic, Liquid Crystal Dispersions. Series on Liquid Crystals; World Scientific: Singapore, 1995; Poulin et al. Science 1997, 275, 1770; Mondain-Monval et al. Eur. Phys. J B 1999, 12, 167). More recently, planar interfaces between thermotropic liquid crystals and aqueous solutions have been used to investigate the orientations of liquid crystals decorated with surfactants (Brake et al. Langmuir 2002, 16, 6101; Brake et al. Langmuir 2003, 16, 6436; Brake et al. Langmuir 2003, 21, 8629), lipids (Brake et al. Science 2003, 302, 2094; Brake et al. Langmuir 2005, 21, 2218), and proteins (Brake et al. Science 2003, 302, 2094). Most recently, the use of a sensor made of multiple grids filled with liquid crystal to detect varying concentrations of LPS in a test sample has been discussed, although the structure of the molecule shown in the paper to be the subject of this study is not LPS nor is it lipid A (McCamley et al. Proc. SPIE 2007, 6441, 64411Y).
There remains a continuing need in the art for new assays for detecting and quantifying LPS at low limits of detection that are specific to LPS and faster than previously disclosed methods.